Tanya Bekyarova

Differential roles of regulatory light chain and myosin binding protein-C phosphorylations in the modulation of cardiac force development

Phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) and myosin binding protein‐C (cMyBP‐C) by protein kinase A (PKA) independently accelerate the kinetics of force development in ventricular myocardium. However, while MLCK treatment has been shown to increase the Ca2+ sensitivity of force (pCa50), PKA treatment has been shown to decrease pCa50, presumably due to cardiac troponin I phosphorylation. Further, MLCK treatment increases Ca2+‐independent force and maximum Ca2+‐activated force, whereas PKA treatment has no effect on either force. To investigate the structural basis underlying the kinase‐specific differential effects on steady‐state force, we used synchrotron low‐angle X‐ray diffraction to compare equatorial intensity ratios (I1,1/I1,0) to assess the proximity of myosin cross‐bridge mass relative to actin and to compare lattice spacings (d1,0) to assess the inter‐thick filament spacing in skinned myocardium following treatment with either MLCK or PKA. As we showed previously, PKA phosphorylation of cMyBP‐C increases I1,1/I1,0 and, as hypothesized, treatment with MLCK also increased I1,1/I1,0, which can explain the accelerated rates of force development during activation. Importantly, interfilament spacing was reduced by ∼2 nm (Δ 3.5%) with MLCK treatment, but did not change with PKA treatment. Thus, RLC or cMyBP‐C phosphorylation increases the proximity of cross‐bridges to actin, but only RLC phosphorylation affects lattice spacing, which suggests that RLC and cMyBP‐C modulate the kinetics of force development by similar structural mechanisms; however, the effect of RLC phosphorylation to increase the Ca2+ sensitivity of force is mediated by a distinct mechanism, most probably involving changes in interfilament spacing.

Protein Kinase A–Mediated Phosphorylation of cMyBP-C Increases Proximity of Myosin Heads to Actin in Resting Myocardium

Protein kinase A-mediated (PKA) phosphorylation of cardiac myosin binding protein C (cMyBP-C) accelerates the kinetics of cross-bridge cycling and may relieve the tether-like constraint of myosin heads imposed by cMyBP-C. We favor a mechanism in which cMyBP-C modulates cross-bridge cycling kinetics by regulating the proximity and interaction of myosin and actin. To test this idea, we used synchrotron low-angle x-ray diffraction to measure interthick filament lattice spacing and the equatorial intensity ratio, I11/I10, in skinned trabeculae isolated from wild-type and cMyBP-C null (cMyBP-C−/−) mice. In wild-type myocardium, PKA treatment appeared to result in radial or azimuthal displacement of cross-bridges away from the thick filaments as indicated by an increase (approximately 50%) in I11/I10 (0.22±0.03 versus 0.33±0.03). Conversely, PKA treatment did not affect cross-bridge disposition in mice lacking cMyBP-C, because there was no difference in I11/I10 between untreated and PKA-treated cMyBP-C−/− myocardium (0.40±0.06 versus 0.42±0.05). Although lattice spacing did not change after treatment in wild-type (45.68±0.84 nm versus 45.64±0.64 nm), treatment of cMyBP-C−/− myocardium increased lattice spacing (46.80±0.92 nm versus 49.61±0.59 nm). This result is consistent with the idea that the myofilament lattice expands after PKA phosphorylation of cardiac troponin I, and when present, cMyBP-C, may stabilize the lattice. These data support our hypothesis that tethering of cross-bridges by cMyBP-C is relieved by phosphorylation of PKA sites in cMyBP-C, thereby increasing the proximity of cross-bridges to actin and increasing the probability of interaction with actin on contraction.

Molecular dynamics of cyclically contracting insect flight muscle in vivo

Flight in insects—which constitute the largest group of species in the animal kingdom—is powered by specialized muscles located within the thorax. In most insects each contraction is triggered not by a motor neuron spike but by mechanical stretch imposed by antagonistic muscles. Whereas ‘stretch activation’ and its reciprocal phenomenon ‘shortening deactivation’ are observed to varying extents in all striated muscles, both are particularly prominent in the indirect flight muscles of insects. Here we show changes in thick-filament structure and actin–myosin interactions in living, flying Drosophila with the use of synchrotron small-angle X-ray diffraction. To elicit stable flight behaviour and permit the capture of images at specific phases within the 5-ms wingbeat cycle, we tethered flies within a visual flight simulator. We recorded images of 340 µs duration every 625 µs to create an eight-frame diffraction movie, with each frame reflecting the instantaneous structure of the contractile apparatus. These time-resolved measurements of molecular-level structure provide new insight into the unique ability of insect flight muscle to generate elevated power at high frequency.

Thick-Filament Strain and Interfilament Spacing in Passive Muscle: Effect of Titin-Based Passive Tension

We studied the effect of titin-based passive tension on sarcomere structure by simultaneously measuring passive tension and low-angle x-ray diffraction patterns on passive fiber bundles from rabbit skinned psoas muscle. We used a stretch-hold-release protocol with measurement of x-ray diffraction patterns at various passive tension levels during the hold phase before and after passive stress relaxation. Measurements were performed in relaxing solution without and with dextran T-500 to compress the lattice toward physiological levels. The myofilament lattice spacing was measured in the A-band (d(1,0)) and Z-disk (d(Z)) regions of the sarcomere. The axial spacing of the thick-filament backbone was determined from the sixth myosin meridional reflection (M6) and the equilibrium positions of myosin heads from the fourth myosin layer line peak position and the I(1,1)/I(1,0) intensity ratio. Total passive tension was measured during the x-ray experiments, and a differential extraction technique was used to determine the relations between collagen- and titin-based passive tension and sarcomere length. Within the employed range of sarcomere lengths (∼2.2-3.4 μm), titin accounted for >80% of passive tension. X-ray results indicate that titin compresses both the A-band and Z-disk lattice spacing with viscoelastic behavior when fibers are swollen after skinning, and elastic behavior when the lattice is reduced with dextran. Titin also increases the axial thick-filament spacing, M6, in an elastic manner in both the presence and absence of dextran. No changes were detected in either I(1,1)/I(1,0) or the position of peaks on the fourth myosin layer line during passive stress relaxation. Passive tension and M6 measurements were converted to thick-filament compliance, yielding a value of ∼85 m/N, which is several-fold larger than the thick-filament compliance determined by others during the tetanic tension plateau of activated intact muscle. This difference can be explained by the fact that thick filaments are more compliant at low tension (passive muscle) than at high tension (tetanic tension). The implications of our findings are discussed.

Myosin binding protein-C phosphorylation is the principal mediator of protein kinase A effects on thick filament structure in myocardium

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) is a regulator of pump function in healthy hearts. However, the mechanisms of regulation by cAMP-dependent protein kinase (PKA)-mediated cMyBP-C phosphorylation have not been completely dissociated from other myofilament substrates for PKA, especially cardiac troponin I (cTnI). We have used synchrotron X-ray diffraction in skinned trabeculae to elucidate the roles of cMyBP-C and cTnI phosphorylation in myocardial inotropy and lusitropy. Myocardium in this study was isolated from four transgenic mouse lines in which the phosphorylation state of either cMyBP-C or cTnI was constitutively altered by site-specific mutagenesis. Analysis of peak intensities in X-ray diffraction patterns from trabeculae showed that cross-bridges are displaced similarly from the thick filament and toward actin (1) when both cMyBP-C and cTnI are phosphorylated, (2) when only cMyBP-C is phosphorylated, and (3) when cMyBP-C phosphorylation is mimicked by replacement with negative charge in its PKA sites. These findings suggest that phosphorylation of cMyBP-C relieves a constraint on cross-bridges, thereby increasing the proximity of myosin to binding sites on actin. Measurements of Ca(2+)-activated force in myocardium defined distinct molecular effects due to phosphorylation of cMyBP-C or co-phosphorylation with cTnI. Echocardiography revealed that mimicking the charge of cMyBP-C phosphorylation protects hearts from hypertrophy and systolic dysfunction that develops with constitutive dephosphorylation or genetic ablation, underscoring the importance of cMyBP-C phosphorylation for proper pump function.

Roles for Cardiac MyBP-C in Maintaining Myofilament Lattice Rigidity and Prolonging Myosin Cross-Bridge Lifetime

We investigated the influence of cardiac myosin binding protein-C (cMyBP-C) and its constitutively unphosphorylated status on the radial and longitudinal stiffnesses of the myofilament lattice in chemically skinned myocardial strips of the following mouse models: nontransgenic (NTG), effective null for cMyBP-C (t/t), wild-type cMyBP-C expressed into t/t (WT(t/t)), and constitutively unphosphorylated cMyBP-C (AllP-(t/t)). We found that the absence of cMyBP-C in the t/t and the unphosphorylated cMyBP-C in the AllP-(t/t) resulted in a compressible cardiac myofilament lattice induced by rigor not observed in the NTG and WT(t/t). These results suggest that the presence and phosphorylation of the N-terminus of cMyBP-C provides structural support and radial rigidity to the myofilament lattice. Examination of myofilament longitudinal stiffness under rigor conditions demonstrated a significant reduction in cross-bridge-dependent stiffness in the t/t compared with NTG controls, but not in the AllP-(t/t) compared with WT(t/t) controls. The absence of cMyBP-C in the t/t and the unphosphorylated cMyBP-C in the AllP-(t/t) both resulted in a shorter myosin cross-bridge lifetime when myosin isoform was controlled. These data collectively suggest that cMyBP-C provides radial rigidity to the myofilament lattice through the N-terminus, and that disruption of the phosphorylation of cMyBP-C is sufficient to abolish this structural role of the N-terminus and shorten cross-bridge lifetime. Although the presence of cMyBP-C also provides longitudinal rigidity, phosphorylation of the N-terminus is not necessary to maintain longitudinal rigidity of the lattice, in contrast to radial rigidity.

Reverse actin sliding triggers strong myosin binding that moves tropomyosin

Actin/myosin interactions in vertebrate striated muscles are believed to be regulated by the “steric blocking” mechanism whereby the binding of calcium to the troponin complex allows tropomyosin (TM) to change position on actin, acting as a molecular switch that blocks or allows myosin heads to interact with actin. Movement of TM during activation is initiated by interaction of Ca2+ with troponin, then completed by further displacement by strong binding cross-bridges. We report x-ray evidence that TM in insect flight muscle (IFM) moves in a manner consistent with the steric blocking mechanism. We find that both isometric contraction, at high [Ca2+], and stretch activation, at lower [Ca2+], develop similarly high x-ray intensities on the IFM fourth actin layer line because of TM movement, coinciding with x-ray signals of strong-binding cross-bridge attachment to helically favored “actin target zones.” Vanadate (Vi), a phosphate analog that inhibits active cross-bridge cycling, abolishes all active force in IFM, allowing high [Ca2+] to elicit initial TM movement without cross-bridge attachment or other changes from relaxed structure. However, when stretched in high [Ca2+], Vi-“paralyzed” fibers produce force substantially above passive response at pCa ∼ 9, concurrent with full conversion from resting to active x-ray pattern, including x-ray signals of cross-bridge strong-binding and TM movement. This argues that myosin heads can be recruited as strong-binding “brakes” by backward-sliding, calcium-activated thin filaments, and are as effective in moving TM as actively force-producing cross-bridges. Such recruitment of myosin as brakes may be the major mechanism resisting extension during lengthening contractions.